Aims: Alloimmune response to red cell transfusion has not been widely investigated in pediatric patients. We retrospectively compared the frequency and specificity of red cell antibody formation among pediatric recipients grouped by age, versus an adult control cohort.

Methods: A total of 331 pediatric red cell transfusion recipients were studied in four age groups: 0 to 4.9 months (Group A), 5.0 to 11.9 months (Group B), 1.0 to 5.9 years (Group C), and 6.0 to 14.9 years (Group D). Similarly transfused adult males, 20.0 to 59.9 years old, as a control cohort group. Alloimmunization was defined as post-transfusion detection of red cell alloantibodies not detected prior to transfusion.

Results: After red cell transfusion, no one in Group A (0 of 106) developed alloantibodies, whereas 8.0% (2 of 25) in Group B, 1.1% (1 of 95) in Group C, and 2.9% (3 of 105) in Group D, versus 2.1% (8 of 380) of adult male controls who developed alloantibodies. However, these differences did not achieve statistical significance.

Conclusion: This investigation of alloimmunization in pediatric recipients found no cases in patients younger than five months old, however, the incidence rates of older age groups were statistically indistinguishable from a control cohort of male adults. Until larger studies suggest otherwise, current antibody screening and cross-matching policies should be continued.

Red blood cell (RBC) alloantibodies, attributable to allogeneic transfusion and pregnancy, are found in 1.43% of screened patients in Japan (1.32% of males and 1.52% of females) [1]. In contrast, neonates and infants up to four months old rarely produce RBC-associated antibodies, neither IgM anti-A and anti-B, nor clinically significant IgG antibodies [2],
[3],
[4],
[5],
[6]. Based on these observations and extensive clinical experience, AABB standards [7] and the British Committee for Standards in Hematology Guideline [8] allow ABO-compatible RBC transfusion without repeated antibody screening/cross-matching if initial antibody screening is negative. Reports of patients under one year old to developing alloantibodies after allogeneic RBC transfusion are few [9],
[10],
[11],
[12],
[13],
[14],
[15]. This retrospective cohort study investigated the frequency and the specificity of RBC alloimmunization among various age groups.

MATERIALS AND METHODS

Pediatric and adult study cohorts
Consecutive pediatric patients transfused at Fukushima Medical University Hospital between January 2003 and December 2015 were enrolled into a retrospective cohort study. Patients included all children younger than 15 years old who received at least one bag of allogeneic packed RBCs for transfusion. RBC transfusions for pediatric patients, including neonates, were matched for ABO and RhD. Neonates received compatible RBCs (usually type O) if they had maternal anti-A and/or anti-B. If a pediatric patient received two or three aliquots from a single RBC bag, we counted one donor exposure. Recipients who were only transfused fresh frozen plasma and/or autologous blood were excluded.

Patients with primary immunodeficiency, any autoimmune disease, those taking immunosuppressive drugs and/or intravenous immunoglobulin (IVIG), and recipients of hematopoietic stem cells and/or solid organ transplants were not excluded from the study. Eligible pediatric patients were grouped by age: 0 to 4.9 months (group A, neonates and younger infants who are rarely alloimmunized), 5.0 to 11.9 months (group B, infants 5 months or older), 1.0 to 5.9 years (group C, preschool children), and 6.0 to 14.9 years old (group D, school children). Adult male patients aged 20 to 59.9 years who received allogeneic RBC during the same study period were used as a control cohort. Those receiving only autologous blood were not included.

All allogeneic blood products are came from Japanese Red Cross Fukushima Blood Center. Japanese Red Cross “units” are based on 200 ml of whole blood, although in modern times 400 ml collections are more common. Whether 200 ml or 400 ml, whole blood is collected in acid-citrate-dextrose solution, made into (packed) RBCs, and stored up to 21 days in Mannitol-Adenine-Phosphate additive solution. Per national Japanese policy, allogeneic cellular blood products are irradiated and all allogeneic blood products, including fresh frozen plasma, are leukoreduced during blood center processing [16].

This study secured approval (#2791) from the Ethics committee of Fukushima Medical University, which is guided by local policy, national law, and the World Medical Association Declaration of Helsinki.

Transfusion testing
RBC alloantibody screening was carried out on patient samples, or, for neonates, maternal samples, using standard physiological saline and polyethylene glycol-indirect antiglobulin test (PEG-IAT) methods
[17],
[18],
[19]. In the physiological saline method, one drop of red blood cell reagent was mixed with two drops (about 100 μL) of patient plasma, and then interpreted after immediate spin (3400 rpm, 15 seconds). PEG-IAT followed by adding 2 drops of an in-house PEG solution to the test tube after physiological saline interpretation, incubating at 37 °C for 15 minutes, and washing by centrifugation four times (Hitachi MC 450, Tokyo, Japan). Then, after adding two drops of anti-human rabbit IgG serum (Ortho Clinical Diagnostics, NJ, USA), it was centrifuged and interpreted. When a test tube showed no reaction, one drop of in-house anti-D-sensitized RBC reagent was added to validate the result.

Surgiscreen and Ortho Diego A (Dia) reagent red cells were used for screening, and Resolve Panel A and Panel B were used for identification of alloantibody specificity (all Ortho Diagnostic Diagnostics).

Outcomes and statistical analysis
The primary outcome of this study was the frequency of alloimmunization among pediatric recipients after RBC transfusion. A secondary outcome was the specificity of newly developed alloantibodies after transfusion.

Statistical analysis was performed using SPSS Statistics version 24 (IBM Analytics, IBM Japan, Tokyo, Japan) and StatMate IV for Windows version 4.01 (ATMS, Tokyo, Japan). The Mann-Whitney U test was used to compare the number of transfused bags between two groups, the Kruskal-Wallis test was used among multiple groups, and antibody positive rates were assessed by the Chi-squared test with p <0.05 regarded as statistically significant.

RESULTS

Patients
As shown in Figure 1, a total of 771 children (428 male, 343 females) met the inclusion criteria, whereas 438 patients were excluded: 242 for no pre-transfusion antibody screening, 5 for positive antibody screening prior to transfusion, and 191 for no post-transfusion antibody screening. Ultimately, 331 were eligible for the study: 106 in group A, 25 in group B, 95 in group C and 105 in group D.

Red cell alloimmunization incidence
Among 331 eligible pediatric recipients, 6 (1.8%, 95% CI: 0.4–3.2%) developed new alloantibodies. Although none of 106 in group A formed alloantibody, 2 of 25 in Group B, 1 of 95 in Group C, 3 of 105 Group D, and 8 of 380 in controls developed new RBC alloantibodies after transfusion. Thus, alloimmunization incidence was 0% [95% CI: 0.0–0.34%] in Group A, 8.0% [95% CI: 0.1–26.0%] in Group B, 1.1% [95% CI: 0.0–5.7%] in Group C, 2.9% [95% CI: 0.3–10.5%] in Group D, 2.1% [95% CI: 0.7–3.5%] among controls (Table 1).

Transfusion characteristics
Among patients under one year old (Groups A and B), transfusion necessitated by congenital disease accounted for 50% or more of cases. On the other hand, from the age of one year and above, blood transfusion was performed more frequently for hematological diseases. The median number of transfused bags was three in Groups A and B, 8 in Group C, and 13 in Group D. Among adult controls, the median was 8 bags. When comparing the number of transfused bags in each group, significant differences (p <0.001) were found in Group A versus Groups C, D and adult controls, and in Group B versus Groups C, D and adult controls. There were also significant differences (p =0.01) in Group C versus Group D, and Group D versus adult controls. The median number of transfused bags by diagnosis were: 2 for low-birth-weight, three each for congenital disease surgery and trauma, 13 for hematological disease, and seven for other diseases. Most RBC components were used for hematological diseases, and significant differences were observed compared with all other conditions.

Details of antibody-producing cases
Table 2 shows clinical details of patients who formed alloantibody. Among six pediatric patients, five were females and only one was a male. In two patients younger than one year old, anti-Jka was detected after latent periods of 49 and 51 days after the first transfusion. The number of latent days until antibody production following the first transfusion ranged from 8 to 51, median of 28.5 days.

The number of transfused bags was 1 to 12, median 3.5. Two cases of antibody production in age Group B are detailed as follows.

A seven-month-old Japanese girl, type O, RhD (-), Ccee, Jk (a-b+), received cross-matched RBCs since five months of life, totaling 7 aliquots of ~140 mL, derived from 4 bags. Anti-E and anti-Jka were detected simultaneously at seven months of age, on the 49th-day after the first transfusion. IVIG was not administered within 5 weeks prior to anti-Jka detection. Hemolysis following RBC transfusion was not observed. Thereafter, the patient was transferred to another hospital, and soon passed away without further RBC transfusion [Case 1].

An eight-month-old Japanese boy, type B, RhD (+), Jk (a-b+), received cross-matched RBCs since six months of life, totaling 4 aliquots of ~140 mL, derived from two bags. Anti-Jka was identified after the last transfusion with no hemolysis. IVIG was not administered prior to antibody detection. Thereafter, he did not need further RBC transfusion. Phenotyping revealed that the first bag was Jk (a-b+), whereas the second bag was Jk (a+b-), and thereafter deemed to be causative. It took 24 days to detect the antibody after the first exposure to the causative antigen [Case 2].

In this study, we found that the overall alloimmunization frequency in pediatric recipients was 1.8% [95% CI: 0.4–3.2%], which was remarkably similar to adult male controls (2.1%, [95% CI: 0.7–3.5%]. As anticipated, the incidence (0%) in Group A (those younger than 5-month-old) was very low compared to older pediatric groups; nevertheless, the differences did not achieve statistically significance. This result is in line with previous reports that no neonates or infants aged four months or less produce RBC-reactive alloantibodies after RBC transfusion
[3],
[4],
[6].

Recently, an interesting German study of 1641 neonates and children up to three years of age reported that only two (0.12%) developed antibodies at ages of 181 and 611 days, which cannot exclude the possibility of “naturally occurring” anti-E and anti-M. The authors concluded that repeat antibody screening and cross-matching during the first months of life can be safely omitted [20]. Our results include two infants aged seven and eight months who formed clinically significant ant-Jka, in contrast to the above work and a previous study in which none of 78 infants up to 11 months old developed antibody [2].

To explain apparent differences in alloantibody production among pediatric recipients between 5.0 and 11.9 months of age, we offer some conjectures. First, genetic background should be considered. We previously observed that none of 52 low-birth-weight Japanese infants who received RBCs - 25 leukoreduced and 27 not, developed anti-HLA at 3 months and 5 to 11 years of age [21]. In contrast, Strauss et al. found that 13% (4/30) of American preterm infants who were multiply transfused with non-leukoreduced RBCs produced white cell antibodies [6]. Moreover, when anti-human leukocyte antigens (HLA) alloimmunization of parous women is compared, a markedly incidence among parous female donors in the USA (31%) is reported
[22], compared to 0.6% of Japanese parous blood donors, or 9.4% of Japanese women currently pregnant when tested by classical lymphocyte toxicity test [23]. As the detection sensitivities of the techniques are markedly different, and Japanese are genetically more homogenous
[24], we cannot draw any conclusions.

Second, we have used standard tube IAT for antibody screening, whereas the German study used anti-human globulin (AHG)-gel column in 12 of 20 years in their study. The latter is less sensitive in detecting weak but clinically significant antibodies compared to standard tube IAT
[19] and solid-phase IAT, especially anti-Jka [25],
[26] Third, effects of donor exposure numbers between the two studies can be excluded, as the median number of donors are 4 in the German study versus 3 among our Group B cohort of patients between 5.0 to 11.9 months of age. Fourth, emergent antibodies might go undetected as a matter of timing. Using mathematical best-fit line equations, Stack and Tormey estimated that only 31.6% of alloantibodies have been detected, owing to antibody testing that is done too early, too late, or not at all [27].

As the alloantibody response in Group C (ages 1.0 to 5.9 years) and Group D (ages 6.0 to 14.9 years) did not differ from adult male recipients, it remains justified to perform pre-transfusion antibody screening and cross-matching in these ages as are done for adults.

In conclusion, pre-transfusion antibody screening is essential for all infants 5.0 months or older, along with post-transfusion follow up. Moreover, it is reasonable to use maternal blood samples for pre-transfusion testing of infants under 5 months of age. Although this single-center study evaluated every eligible case over 13-year interval, and no patients under 5 months of age developed red cell alloantibodies, statistical power was insufficient to justify curtailing current testing strategies. We anticipate a prospective, multicenter design with large number of recipients will inform future practices.

Author Contributions
Maiko Abe – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Hitoshi Ohto – Substantial contribution to conception and design, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Keiji Minakawa – Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Final approval of the version to be published
Kinuyo Kawabata – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Satoshi Ono – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Nozomi Takano – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Hiroe Suzuki – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Mao Watanabe – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Akiko Sugawara – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Masami Kikuchi – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Saori Miura – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Mao Watanabe – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Chikako Takeuchi-Baba – Substantial contribution to acquisition of data, Drafting the article, Final approval of the version to be published
Hiroyasu Yasuda – Substantial contribution to conception and design, Acquisition of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Kenneth E. Nollet – Substantial contribution to interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Yoshiko Tamai – Substantial contribution to conception and design, Drafting the article, Final approval of the version to be published
Junichi Kitazawa – Substantial contribution to conception and design, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Kazuhiko Ikeda – Substantial contribution to conception and design, Drafting the article, Final approval of the version to be published

Guarantor of Submission
The corresponding author is the guarantor of submission.

Source of Support
None

Consent Statement
Written informed consent was obtained from patients prior to transfusion.